The skin is made up of two layers: epidermis and dermis. The outer layer is the epidermis which is made up mainly of keratinocytes, melanocytes and Langerhans cells and its basic function is to retain body water, act as a barrier against harmful chemical agents as well as against pathogen agents, and perform cellular renovation processes. The inner layer, dermis, formed by fibroblasts, adipocytes and macrophages is tightly connected to the epidermis through the basal membrane and it contains numerous nerve endings which provide tactile and temperature sensations. It also houses hair follicles, sweat glands, sebaceous glands, Apocrine Glands and blood vessels, and one of its main functions is to keep the skin elasticity and appearance.
The dermis also includes the extracellular matrix, formed by a group of extracellular proteins (fibrous proteins, glycoproteins and proteoglycans) whose principal function is to keep skin structure. Correct tissue functioning and development depend on the right formation of the extracellular matrix and on the right regulation of its components [Wiberg C., Klatt A. R., Wagener R., Paulsson M., Bateman J. F., Heinegard D. and Morgelin M. (2003) “Complexes of matrilin-1 and biglycan or decorin connect collagen VI microfibrils to both collagen II and aggrecan” J. Biol. Chem. 278:37698-37704]. The two most important fibrous proteins in the extracellular matrix are collagen and elastin, which are responsible for the mechanical properties of the tissues such as the ability to resist tension, compression, extensibility and torsion. Proteoglycans have a structural and metabolic function, while glycoproteins, together with proteoglycans, work as a union bridge between matrix components and cells [Aumailley M. and Gayraud B. (1998) “Structure and biological activity of the extracellular matrix” J. Mol. Med. 76:253-265; Culav E. M., Clark C. H. and Merrilees M. J. (1999) “Connective tissues: matrix composition and its relevance to physical therapy” Phys. Ther. 79:308-319; Scott J. E. (2003) “Elasticity in extracellular matrix ‘shape modules’ of tendon, cartilage, etc. A sliding proteoglycan-filament model” J. Physiol. 553:335-343].
Collagens are a family of fibrous proteins of the extracellular matrix that constitute a 25% of the total proteic mass in mammals. They have been classified in more than 20 families, all of them having individual characteristics which fulfill specific functions in different tissues.
The main characteristic of collagen is its helicoidal structure formed by the association of three polypeptide chains rich in glycine and proline. Alterations in its aminoacid composition cause dysfunction and loss of its mechanical properties [Culav E. M., Clark C. H. and Merrilees M. J. (1999) “Connective tissues: matrix composition and its relevance to physical therapy” Phys. Ther. 79:308-319]. These polypeptide chains can associate one to the other and form fibrils, which have a diameter of 10-300 nm and a length of up to hundreds of micrometers in mature tissues. These fibrils are often added into major structures, such as cable bunching, which can be seen through electronic microscopy as collagen fibers of many micrometers in diameter. This process is known as fibrillogenesis [Aumailley M. and Gayraud B. (1998) “Structure and biological activity of the extracellular matrix” J. Mol. Med. 76:253-265]. Not all collagens have the ability to form fibrils; only I, II, III, V and XI type collagens, which are known as fibrillar collagens.
An adult dermis is basically formed by fibrillar collagens type I, III and V. Type I collagens represent 80-90% of the total collagen of the dermis. Generally, type I collagen fibers feature a bigger diameter, which correlates with its ability to withstand a bigger mechanical load. Type III collagen intervenes in tissue extensibility, and as years go by, it is replaced by type I collagen molecules, process which is partly responsible for mature skins being less extensible than young skins. Type V collagen associates with types I and III regulating the diameter of fibrils [“The Biology of the Skin”, Freinkel R. K. and Woodley D. T, eds. The Parthenon Publishing Group, 2001; Culav E. M., Clark C. H. and Merrilees M. J. (1999) “Connective tissues: matrix composition and its relevance to physical therapy” Phys. Ther. 79:308-319].
Collagen fibers are in constant renewal process, but such renewal decreases with age, causing the thinning of dermis. Besides, even though collagen fibers organization provides collagen network with great resistance, collagen fibers are sensitive to certain enzymes known as matrix metalloproteases (MMP). MMPs belong to a family of proteolytic enzymes (endoproteases) which contain a zinc atom coordinated with three cysteine residues and one residue of methionine in its active center and which can, collectively, degrade macromolecular components from the extracellular matrix and from the basal laminas into a neutral pH (collagen, elastin, etc.).
The family of matrix metalloproteases is classified according to its structural similarity and its substrate specificity [Woessner J. F. (1991) “Matrix metalloproteinases and their inhibitors in connective tissue remodeling” Faseb J. 5:2145-2154; Miyazaki K. and Higashi S. (1996) “Matrix metalloproteinases: their structures and functions, with special reference to their roles in tumor invasion and metastasis” Seikagaku 68:1791-1807]. Within the family of MMPs there are collagenases which degrade fibrilar collagen (MMP-1 or interstitial collagenase, MMP-8 or neutrophil collagenase, MMP-3 or collagenase 3), gellatinases which degrade type IV collagen or any other form of denaturalized collagen (MMP-2 or gellatinase A 72 kDa and MMP-9 or gellatinase B 92 kDa), stromelysins whose wide spectrum of activity is directed to the extracellular matrix proteins such as glycoproteins like fibronectin or laminin and proteoglycans, among others (MMP-3 or stromelysin 1, MMP-10 or stromelysin 2 and MMP-11 or stromelysin 3), matrilysin (MMP-7) metalloelastase (MMP-12) or the membrane metalloproteases (MMP-14, MMP-15, MMP-16 and MMP-17).
Metalloproteases are produced and secreted in an inactive way (proenzyme), which is later activated in the extracellular environment by the loss of the propeptide region of its sequence. The members of this protein family can activate one another. The MMP activity regulation can take place in different ways: regulating gens expression (transcription and transfer), regulating inactive process activation or acting locally on the active process.
MMPs play an important role in different skin, mucosae and/or scalp conditions and disorders in which there is a degradation and destruction of extracellular proteins [Kahari V. M. and Saarialho-Kere U. (1997) “Matrix metalloproteinases in skin” Exp. Dermatol. 6:199-213]. Among the different pathologies described in which there is a MMP overexpression or an increase of MMP activity in connective tissue cells, we find chronic ulcer [Miyoshi H., Kanekura T., Aoki T. and Kanzaki T. (2005) “Beneficial effects of tissue inhibitor of metalloproteinases-2 (TIMP-2) on chronic dermatitis” J. Dermatol. 32:346-353], psoriasis [Flisiak I., Mysliwiec H. and Chodynicka B. (2005) “Effect of psoriasis treatment on plasma concentrations of metalloproteinase-1 and tissue inhibitor of metalloproteinase-1” J. Eur. Acad. Dermatol. Venereol. 9:418-421; Suomela S., Kariniemi A. L., Impola U., Karvonen S. L., Snellman E., Uurasmaa T., Peltonen J., Saarialho-Kere U. (2003) “Matrix metalloproteinase-19 is expressed by keratinocytes in psoriasis” Acta Derm. Venereol. 83:108-114], oral pathologies such as gingivitis and periodontitis [Reynolds J. J. and Meikle M. C. (1997) “The functional balance of metalloproteinases and inhibitors in tissue degradation: relevance to oral pathologies” J. R. Coll. Surg. Edinb. 42:154-160], skin cancer [Ntayi C., Hornebeck W and Bernard P. (2004) “Involvement of matrix metalloproteinases (MMPs) in cutaneous melanoma progression” Pathol. Biol. (Paris) 52:154-159; Kerkela E. and Saarialho-Kere U. (2003) “Matrix metalloproteinases in tumor progression: focus on basal and squamous cell skin cancer” Exp. Dermatol. 12:109-125] and tumor invasion and metastasis [Sato H., Takino T. and Miyamori H. (2005) “Roles of membrane-type matrix metalloproteinase-1 in tumor invasion and metastasis” Cancer Sci. 96:212-217].
MMPs also play a key role in different physiological situations in which the extracellular matrix is degraded or reconstructed, such as the extracellular matrix proteolytic remodeling, including tissue morphogenesis during development, tissue repair and angiogenesis [Kahari V. M. and Saarialho-Kere U. (1997) “Matrix metalloproteinases in skin” Exp. Dermatol. 6:199-213]. In a particular way, MMPs have a crucial role in connective tissue remodeling [Abraham D., Ponticos M. and Nagase H. (2005) “Connective tissue remodeling: cross-talk between endothelins and matrix metalloproteinases” Curr. Vasc. Pharmacol. 3:369-379], for example collagen degradation by MMPs makes the skin look wrinkled and flaccid.
Likewise, MMPs participate in skin aging. Different factors, including exposure to ultraviolet (UV) radiation, produce collagen degradation, with all the consequences it entails on skin structure and/or firmness, particularly on those skin areas exposed to the solar light such as the face, ears, neck, scalp, arms and hands.
Skin damage associated to chronic exposition (repetitive irradiation) or high exposition (strong irradiation) to UVA and/or UVB rays has been studied; particularly it is known that                UVB rays (290-300 nm; 5% of total UV rays) with more energetic wavelength, especially affect epidermic cells (keratinocytes) acting over its DNA.        UVA rays (320-400 nm; 95% of total UV rays) have a stronger penetration grade and also act over dermic cells such as fibroblasts and they act indirectly generating free radicals.        
Moreover, prolonged exposure to UV radiation, particularly to UVA and/or UVB radiation stimulates MMP expression [Fisher G. J., Datta S. C., Talwar H. S., Wang Z. Q., Varani J., Kang S. and Voorhees J. J. (1996) “Molecular basis of sun-induced premature skin ageing and retinoid antagonism” Nature 379:335-339; Fisher G. J., Wang Z. Q., Datta S. C., Varani J., Kang S. and Voorhees J. J. (1997) “Pathophysiology of Premature Skin Aging Induced by Ultraviolet Light” New Eng. J. Med. 337:14191429; Fisher G. J., Choi H. C., Bata-Csorgo Z., Shao Y, Datta S., Wang Z. Q., Kang S. and Voorhees J. J. (2001) “Ultraviolet irradiation increases matrix metalloproteinase-8 protein in human skin in vivo” J. Invest. Dermatol. 117:219-226], especially matrix metalloelastase type 1 (MMP-1). This is one of the components of photoinduced skin aging (or photoaging) [Rittie L. and Fisher G. J. (2002) “UV-light-induced signal cascades and skin aging” Ageing Res. Rev. 1:705-720]. Besides, it is known that MMP-1, MMP-2 and MMP-9 activity increases with age and that this increase, together with cell growth deceleration, contributes to chronologic skin aging [EP 1 005 333 B1]. Similarly, smokers' skin also has a premature aging aspect in which MMPs are overexpressed [Lahmann C., Bergemann J., Harrison G. and Young A. R. (2001) “Matrix metalloproteinase-1 and skin aging in smokers” Lancet 357:935-936].
Another skin and/or scalp pathologies or disorders, associated to MMP overexpression or to an increase of MMP activity in the connective tissue is acne [Papakonstantinou E., Aletras A. J., Glass E., Tsogas P., Dionyssopoulos A., Adjaye J., Fimmel S., Gouvousis P., Herwig R., Lehrach H., Zouboulis C. C. and Karakiulakis G. (2005) “Matrix metalloproteinases of epithelial origin in facial sebum of patients with acne and their regulation by isotretinoin” J. Invest. Dermatol. 125:673-684]. It is described that skins affected by acne have high levels of MMP-1.
Likewise, rosacea is a skin and/or scalp pathology or disorder in which MMPs are also involved. Rosacea is characterized by an increase of angiogenesis and inflammation. Angiogenesis refers to the process of new blood vessels formation and it includes benign conditions such as rosacea and malignant processes such as cancer. Matrix degrading enzymes, present in tissue extracellular matrix facilitate angiogenesis since they allow new blood vessels to penetrate the matrix. MMPs represent a kind of enzymes involved in such processes [Sapadin A. N., Fleischmajer R. (2006) “Tetracyclines: Nonantibiotic properties and their clinical implications” J. Am. Acad. Derm. 54:258-265].
People with dermatitis, including contact dermatitis and atopic dermatitis, also have high levels of some MMPs [Herouy Y, Mellios P., Bandemir E., Dichmann S., Nockowski P., Schöpf E. and Norgauer J. (2001) “Inflammation in stasis dermatitis upregulates MMP-1, MMP-2 and MMP-13 expression” J. Dermatol. Sci. 25:198-205; Devillers A. C., van Toorenenbergen A. W., Klein Heerenbrink G. J., Muldert P. G. and Oranje A. P. (2007) “Elevated levels of plasma matrix metalloproteinase-9 in patients with atopic dermatitis: a pilot study” Clin. Exp. Dermatol. 32:311-313; Miyoshi H., Kanekura T., Aoki T. and Kanzaki T. (2005) “Beneficial effects of tissue inhibitor of metalloproteinases-2 (TIMP-2) on chronic dermatitis” J. Dermatol. 32:346-353]. “Dermatitis” is defined as those skin conditions, disorders or pathologies that cause inflammation, including contact dermatitis, atopic dermatitis, sensitive skin and eczema. It is also known that MMPs are involved in perifollicular matrix degradation, and thus, in hair loss. Specifically, cytokines and the epidermal growth factor stimulate MMP-9 production in the lower epithelial compartment of hair root, such mechanism controls capillary follicle involution observed in alopecia [Jarrousse F., Boisnic S., Branchet M. C., Beranger J. Y., Godeau G., Breton L., Bernard B. A. and Mahé Y. F. (2001) “Identification of clustered cells in human hair follicle responsible for MMP-9 gelatinolytic activity: consequences for the regulation of hair growth” Int. J. Dermatol. 40:385-392]. Thus, overexpressed MMP inhibition during alopecic processes could be effective in delaying, and even preventing, hair loss [EP 1 076 549 B1].
Also, MMP activity is related to scar formation in tissues containing collagen. “Scar formation is defined as the formation of an abnormal morphological collagen structure due to previous injuries or due to the healing process of tissue containing collagen on the skin.
Healing processes consist of three stages: (1) inflammation, (2) tissue formation and (3) tissue remodeling. A necessary stage in the healing process is extracellular matrix degradation: in order for the cells to proliferate in the wounded area and regenerate it, it is necessary that the extracellular matrix be degradated. Such degradation is made through MMPs. Healing process stages are regulated by a balance between the different MMPs and it has been described that an excess of MMP activity causes chronic ulcers. For example, an overexpression of MMP-8 can be associated to the pathogenesis of leg chronic ulcers. Likewise, diabetic ulcers are characterized by a prolonged inflammation, decrease collagen synthesis and high MMP levels.
Most scars consist of collagen fibers irregularly organized as well as an excess of collagen. Scars have different causes (accidents, surgery, skin diseases, burns, acne, infections and accidents in general), but not all scars are the same. Different kinds of scars can be grouped in                Flat and pale scars: formed as a result of the body's natural healing process.        Sunken scars: formed by skin attached to deeper structures, such as muscles, or due to loss of fat in internal tissues. These scars are recessed into the skin and are usually the result of an injury.        Hypertrophic scars: appear when the body produces an excess of collagen during the healing process. These scars elevate over the skin surface and contain irregularly organized collagen.        Keloid scars: formed as a result of an imbalance in the production of collagen during the healing process. These scars not only elevate over skin surface, but also they extend beyond the boundary of the original wound and can continue to grow indefinitely.        Acne scars: formed in skin affected by acne. The scar can be sunken or become a keloid. People who have had chicken pox can have similar scars.        Stretched scars: occur when the skin around a healing wound is put under tension during the healing process. Initially, the scar may appear normal but can widen and thin over a period of weeks or months. This can occur when the wound is close to a joint and is stretched during movement or it may be due to poor healing because of general ill health or malnutrition.                    Stretch marks: develop when the skin is stretched rapidly, for example during pregnancy or the adolescent growth spurt.                        
Therefore, skin scar reduction is desirable both from the pathological point of view, as healing during fibrotic processes, and from the cosmetic point of view, as in the case of softening the aspect of scars caused by acne or stretch marks.
It has also been described that during adipocytes proliferation and differentiation, MMPs are overexpressed [Traurig M. T., Permana P. A., Nair S., Kobes S., Bogardus C. and Baier L. J. (2006) “Differential expression of matrix metalloproteinase 3 (MMP3) in preadipocytes/stromal vascular cells from nonobese nondiabetic versus obese nondiabetic Pima Indians” Diabetes 55:3160-3165]. MMP activity inhibition with several specific inhibitors prevents adipocytes differentiation. An especially interesting fact is that MMP inhibitors are able to reduce the accumulation of lipogenic markers (triglycerides) in adipocyte cultures [Demeulemeester D., Collen D. and Lijnen H. R. (2005) “Effect of matrix metalloproteinase inhibition on adipose tissue development” Biochem. Biophys. Res. Commun. 329:105-110]. Thus, MMP inhibitors can be developed as anti cellulite agents and help reduce orange peel skin aspect.
MMP activity is also responsible for the extracellular matrix disorganization that surrounds lymphatic and blood vessels. Matrix deterioration around blood vessels allows for a passive vasodilatation which gives place to capillary visibility or telangiectasia, or couperosis. Besides, this microcapillary passive dilatation can cause local blood vessel bursts which can give place to bags under the eyes or dark circles in the periorbital area. Furthermore, MMPs have an influence over vein wall mechanical properties, which can make veins fragile and consequently lead to the development of varicose veins.
Apart from the relation of MMPs to tissue matrix degradation, it has been suggested that MMPs are also involved in different pathologies that concur with an abnormal metabolism of the connective tissue or basal membrane matrix such as arthritis (rheumatoid arthritis, osteoarthritis, etc), bone diseases (osteoporosis, etc.), ectopic angiogenesis, multiple sclerosis, tumors metastasis and tissue ulcers (cornea, stomach, epidermis, etc.) [EP 0 927 161 B1]. Therefore, an MMP inhibitor could be effective in treating and preventing those pathologies caused by an abnormal metabolism of the tissular matrix.
Then, it is widely accepted that MMP activity regulation is highly important for the basal membrane and extracellular matrix protection, as well as for preventing and improving signs of aging. In the context of the present invention, the term “aging” refers to changes experienced by the skin with the passing of years (chrono-aging), or due to sun exposition (photoaging) or due to environmental agents like tobacco smoke, extreme cold or wind weather conditions, chemical pollutants or pollution and it includes all visible external changes as well as those perceptible by touch, such as for example and in a non-limiting sense, development of skin discontinuities such as wrinkles, thin lines, cracks, irregularities or roughness, increase of pore size, loss of elasticity, loss of firmness, loss of smoothness, loss of the capacity to recuperate after deformation, skin hanging such as cheek hanging, appearance of eye pouches or double chin, among others, changes of the skin color, such as marks, reddening, bags under the eyes or the appearance of hyperpigmented areas such as age marks or freckles among others, anomalous differentiation, hyperkeratinization, elastosis, keratosis, orange-peel skin, loss of collagen structuring and other histological changes of the stratum corneum, of the dermis, epidermis, vascular system (for example the appearance of spider veins or telangiectasias) or of those tissues close to the skin, among others.
The cosmetic and pharmaceutical sectors have identified numerous compounds and plant extracts which are effective as MMP inhibitors and there are different bibliographical revisions in literature about MMPs, pathologies associated to their overexpression or their activity increase and the different families of compounds and plant extracts useful to their inhibition. In the state of the art there have been described different approximations to control MPPs activity, including small molecules [Levy D. E., Lapierre E., Liang W, Ye W, Lange C. W, Li X., Grobelny D., Casabonne M., Tyrrell D., Holme K., Nadzan A. and Galardy R. E. (1998) “Matrix metalloproteinase inhibitors: A structure activity study” J. Med. Chem. 41:199-223; Wojtowicz-Praga S. M., Dickson R. B. and Hawkins M. J. (1997) “Matrix metalloproteinase inhibitors” Investigational new Drugs 15:61-75; Duivenvuurden W. C. M., Hirte H. W. and Singh G. (1997) “Use of tetracycline as an inhibitor of matrix metalloproteinase activity secreted by human bone metastasizing cancer cells” Invasion and Metas. 17:312-322] peptidic inhibitors [Odake S., Monta Y. and Morikawa T. (1994) “Inhibition of matrix metalloproteinases by peptidyl hydroxamic acids” Biochem. Biophys. Res. Comm. 199:1442-1446] or antibodies against MMPs [Su J-L., Becherer D., Edwards C., Bukhart W, McMgeehan G. M. and Champion B. R. (1995) “Monoclonal antibodies against human collagenase and stromelysin” Hybridoma 14:383-390]. Cosmetic industry has made important efforts to offset MMPs activity and the age-related loss of functionality of extracellular matrix components caused by MMPs. Balance between production and degradation of skin essential biomolecules such as collagen evolves with aging towards degradation processes, which leads to, for example, a progressive thinning and disorganization of the dermis which produces dermis flaccidity and a subsequent formation of wrinkles. Therefore, those methods which allow to delay or prevent extracellular matrix degradation will have a potential beneficial effect on mature skins or on aged and/or photo-aged skins; allowing them to partially recover the mechanical properties (elasticity, flexibility and firmness) which they have lost due to age or sun exposure and/or environmental pollutants and thus show a better appearance with fewer wrinkles and a smoother skin. Likewise, MMP inhibition is also an important aspect for the cosmetic sector for applications other than delaying the aging and/or photo-aging, such as for example hair growth modulation [EP 1 076 549 B1] or wound treatments [US 2004/0127420 A1; US 2003/0166567 A1].
Despite the great number of existing compounds and/or extracts, there is still a need to identify new more effective and selective MMP inhibitors.
In the present invention there are described peptides which are effective in MMP inhibition, imitating on this way the function of endogenous MMP inhibitors (TIMP, matrix metalloproteinase tissue inhibitor). The peptide sequence of the invention is not contained in proenzymatic MMP sequences, such as the peptide sequences described in US 2004/0127420 A1 and US 2003/0166567 A1. Sequences similar to the peptides of the invention, without the citrulline residue on the carboxy-terminal (C-terminal), are found in sequences of different enzymes or have enzymatic activity [WO 2004/033668 A2; WO 99/00489 A1]; there is not any clue in the state of the art that suggests the effectiveness of the peptides of the invention as MMP inhibitors, so a person skilled in the art could not deduce the nature of the peptides which inhibit MMPs.